I am new to the forums, but I have been a longtime lurker when the need arose. I am having some difficulty cloning a 1kb insert into the pEGFP vector (Kan resistant).

I know my ligated product is present via PCR and by simply running it on a gel. My problem is when I transform my plasmid, I get zero colonies. I have tried multiple methods of transformation with a variety of cells. Commercially competent one shot, sure2, XL-10 gold, able k, and also lab produced DH5alpha and DH10Beta. I will usually use 50-100ul of cells with 1-5uL of ligated product (depending on concentation, with max being 50ng). I will allow cells to incubate for 30min, heat shock for 45 sec at 42C (or commercial competent suggested duration/temp) and allow cells to recover for 2-5 min on ice. I then rotate cells at 37C for 1-3 hrs and plate.

can you do a double digest to re-confirm your insert is there? I have worked with N2 and N3, and your protocol looks fine to me. One thing that I suspect is that your insert itself could be toxic? or your competent cells are not viable enough due to a possible break down of your freezer. Trust me this has happened to us like more than 5-6 times that during weekend we had no electricity and nobody knew about it. These cells might grow in broth again, but are not suitable for transformation anymore. Kan plates also last for long time, unlike amp plates that can't be used after a month.

I have done a digest for both original insert cloning sites and a digest in the middle of my insert and random plasmid RE site. Both show the expected size. I have also cloned my insert in other plasmids, so I am fairly certain my insert is not toxic to e.coli. I checked the competency of my cells with pUC19 and everything seems normal. Also freezers are relayed to a central computer. Any problems would send an email to our PI. This is really troubling for me. Do you suggest anything else?

Sticky ends. I am using 25ug/mL Kanamycin (prepared recently). I have done controls of my N-1/C-1 vector (both produce colonies), checking my linearized vector to see if plasmid re-ligated, shows zero colonies. I am using BglII and PstI, also using SalI and BamHI.

Incubator itself is fine. Other members are using same equipment and are seeing positive results.

I grow my E.coli for 1-4 hrs at 37C, while rotating. I then remove ~150uL of cells and plate. I then spin down cells at 4,000xg for 1min and remove supernatant. I add 150uL of LB, resuspend pellet and plate mixture. I have used this protocol many times and have been successful at obtaining positive clones.

I am new to the forums, but I have been a longtime lurker when the need arose. I am having some difficulty cloning a 1kb insert into the pEGFP vector (Kan resistant).

I know my ligated product is present via PCR and by simply running it on a gel. My problem is when I transform my plasmid, I get zero colonies. I have tried multiple methods of transformation with a variety of cells. Commercially competent one shot, sure2, XL-10 gold, able k, and also lab produced DH5alpha and DH10Beta. I will usually use 50-100ul of cells with 1-5uL of ligated product (depending on concentation, with max being 50ng). I will allow cells to incubate for 30min, heat shock for 45 sec at 42C (or commercial competent suggested duration/temp) and allow cells to recover for 2-5 min on ice. I then rotate cells at 37C for 1-3 hrs and plate.